ESHG Posters 24

L. Abbasi Moheb¹, A. Hatami¹, M. R. Seddighi Gilani², P. Afsharian², S. Mollah Mohammadi¹, K. Javan¹, H. Najmabadi¹;
¹Genetics Research Center the Social Welfare and Rehabilitation University, Tehran, IRAN (ISLAMIC REPUBLIC OF), ²Department of Andrology and Clinical and Experimental Genetics,Royan Institute, Tehran, IRAN (ISLAMIC REPUBLIC OF).

Male factor is the cause of infertility in about 50 percent of infertile couples. 3-20% of infertile men with severe oligozoospermia or azoospermia have microdeletions of the long arm of Y chromosome (Yq) consistent with the location of AZF (Azoospermia Factor) in Yq11.23.
Recent Studies have shown the presents of 4 regions on the interval 6 of Y choromosome associated with male infertility. These are AZFa, AZFb, AZFc and AZFd which are involved in the process of spermatogenesis. DAZ and RBM are two multicopy gene families, which are expressed only in testis and have an important role in spermatogenesis. They are located at AZFc and AZFb respectively. Microdeletions in these regions cause severe oligozosspermia or azoospermia.
In this study DNA was extracted from blood lymphocytes of 120 azoospermic or severe oligozoospermic men in whom all known causes of infertility had been excluded. Twenty one Y specific STSs of deletion intervals 5 and 6 were selected for typing all subjects.
Of the 120 oligo/azoospermic men, 8 (6.6%) had deletions of more than one STSs. All of the patients had microdeletions in AZFc (interval 6D), in which 75% of the cases had deletion in DAZ gene. Fifty percent of these individuals their deletion extended in AZFb region (Interval 6A).No microdeletions were detected in AZFa and RBM.

Clinical, cytogenetic and molecular genetic findings in patients with sterility or repeated pregnancy loss.

R. Gaillyová¹, P. Kuglík², A. Oltová¹, B. Ravèuková¹, J. Žáková³;
¹Department of Medical Genetics, University Hospital, Jihlavská 20, 639 00, Brno, CZECH REPUBLIC, ²Faculty of Science, Masaryk University, Kotláøská 2, 611 37, Brno, CZECH REPUBLIC, ³Centre of Assisted Reproduction,1st Clinic of Gynaecology/Obstetrics, University Hospital, Jihlavská 20, 639 00, Brno, CZECH REPUBLIC.

In our study, cytogenetic and molecular genetic analyses were performed in 305 patients with sterility or repeated pregnancy loss from the Centre of Assisted Reproduction.
Cytogenetic examinations using karyotyping of peripheral blood were performed in all 305 individuals. Chromosomal abnormalities (mainly numerical aberrations of gonosomes, balanced translocations or inversions) were detected in about 10% patients.
DNA analyses were performed in two groups of 214 patients with reproductive failures. In the first group of patients (infertile men with oligo- or azoospermia) we performed molecular analyses of the Y by the PCR of sequences-tagged sites (STS-PCR) and mutation analyses of the most common mutations in the CFTR gene. In the second group of patients (positive family history or repeated pregnancy loss) we studied Leiden mutations of the factor V gene (G1691A) and the most common CFTR mutations. In all tested patients we diagnosed 7 carriers of CFTR mutations (dF508, dele 2,3(21kb), G542X), 7 carriers of Leiden mutations G1691A (1q23) and 4 men with the microdeletion of AZFc region on Yq chromosome.
Our results of the complex screening programme in couples with sterility or repeated pregnancy loss from the Moravia region demonstrate chromosomal and / or gene disorders in 15,4% of patients.
In 8 patients with repeated IVF failures or spontaneous abortion we performed preimplantation genetic diagnosis (PGD) of numerical abnormalities for chromosomes 13, 18, 21, X and Y using fluorescence in situ hybridization (FISH). Two of the women have become pregnant after PGD.

R. Behulová¹, R. Mezenská¹, R. Weisenpacherová¹, M. Lukácová¹, J. Simko²;
¹Centre of Medical Genetics, University Hospital, Bratislava, SLOVAKIA, ²2-nd Gynecologic-Obsteric Clinic, Komensky University, Bratislava, SLOVAKIA.

Male factor infertility accounts for about half of the cases of couple infertilty. Microdeletions of the long arm of the human Y chromosome are associated with spermatogenic failure and have been used to define three regions of Yq (AZFa, AZFb, AZFc) that are recurrently deleted in infertile males. Several genes have been identified within this region and have been proposed as candidates for infertility. About 10-15% of azoospermic and about 5-10% of severely oligospermic men have Yq microdeletions. The deletions are associated with a wide range of histological pictures ranging from Sertoli cell only syndrome to spermatogenic arrest and severe hypospermatogenesis. Assisted reproduction techniques such as in vitro fertilization and Intra Cytoplasmic Sperm Injection alone represent an efficient therapy for these patients. However the potencial of these techniques to transmit genetic defects causing male infertility raises the need for a systematic genetic screening and genetic counselling of these patients.
We have investigated 168 infertile men (67 with azoospermia, 61 with oligoasthenospermia, 26 with oligospermia and 11 with other diagnosis). For each patient, five multiplex PCR analyses were performed on DNA isolated from leukocytes derived from peripheral blood to screen 10 sY- sequences on Yq. Microdeletions were detected in 11/168 (6,5%) infertile men. Seven of the 67 azoospermic men (10,7%) had such deletions. Of the 61 oligoasthenospermic men 2 (3,2%) had deletion, 1 of the 26 oligospermic man had deletion, and among other diagnoses microdeletion was detected in 1 man.

Molecular analysis of the Yq microdeletions in male with azoospermia and oligospermia and in children of liquidators of Chernobyl accident consequences from Ukraine.

O. A. Yasinskaya¹, S. G. Malyarchuk¹, V. M. Zinchenko², L. A. Livshits¹;
¹Institute of Molecular Biology and Genetics, Kiev, UKRAINE, ²"ISIDA-IVF" clinic, Kiev, UKRAINE.

Male infertility is caused by many different exogenous and endogenous factors. In addition to chromosomal anomalies, microdeletions in the azoospermic factor region (AZF) in the long arm of Y-chromosome have been detected in men with azoospermia or sever oligospermia. We have screened 105 men with azoospermia and oligospermia - patients of “ISIDA-IVF” clinic involved in ICSI (intracytoplasmic sperm injection) program as well 70 boys - sons of fathers-liquidators (Chernobyl eccident clean-up workers). Our study consists of 11 primer pairs that are homologous to previously identified and mapped sequence tagged sites (STS). The STS primers tested on each subject were sY84, sY85 (AZFa); sY117; sY124, sY134 (AZFb); sY141, sY146; sY240, sY254 (DAZ), sY255 (DAZ), sY158 (AZFc). The samples were analysed for Y-chromosome microdeletion by multiplex-PCR. The PCR products were analyzed on a 1,8% agarose gel. SRY was used as internal controls of PCR reactions. Any de novo deletions was not found in the group of liquidators children. In five of the 105 infertil men (4,76%) it was shown 1 deletion of at AZFb (0,95%) and 4 deletions of at AZFc (gene DAZ) (3,81%) regions prevailing which majority is concentrated in AZFc region. During patients research we have made a conclusions: a) the damage of genes from AZFb region results in impairments of last stage spermatogenesis; b) deletions in AZFc region are critical for early stage spermatogenesis. The genetic consultating and preimplantation sex analysis were recommended for the patients with found deletions.

Dicentric chromosome (Y;22) resulting in a microdeletion encompassing SHOX in a boy with short stature and no dysosteochondrosis.

C. Borie¹, J. Léger¹, O. Dupuy¹, A. Lebbar², C. Pignal¹, A. Thaly¹, M. Vitu¹, P. Czernichow¹, P. L. Eydoux¹;
¹Hôpital Robert Debré, Paris, FRANCE, ²Hôpital Cochin, Paris, FRANCE.

We report a 4 year-old child with short stature, and a dicentric chromosome with a deletion of the distal part of chromosome Yp. The parents were not consanguineous, and did not have any relevant history of genetic disease. The pregnancy was uneventful, until intra-uterine growth retardation was noted. Prenatal karyotyping showed a (Y;22) translocation. No structural fetal abnormality was shown at ultrasound examination, and the pregnancy went to term. A growth retarded boy with an otherwise normal physical examination was delivered at 39 weeks. At age 4, the child had short stature (-3SD) without mental retardation. Radiological examination of the wrist was normal. Blood karyotyping confirmed the chromosomal rearrangement. C-banding showed a dicentric chromosome, and FISH with centromeric probes confirmed the presence of both chromosome Y and 22 centromeres on the derivative chromosome. FISH using a TUPLE1 probe did not show any rearrangement at 22q11 or at the ARSA locus. Using a subtelomeric probe, a deletion of the distal Yp region was shown, whereas SRY was not deleted as shown by FISH. A set of two probes encompassing the SHOX region were used for FISH and demonstrated a deletion of this gene. The karyotype was thus 45,X,der(Y;22)(p11;q11).ish del(Y)(p11p11) (SRY+,SHOX-).
Haploinsufficiency of SHOX may result in isolated growth retardation or in Leri-Weill dysostosis, which is not an obvious diagnosis in our patient, but may appear at a later age. Although other loci may have been deleted, no other genes seem to have resulted in haploinsufficiency in our patient.

Cytogenetic Analysis And Y Chromosome Microdeletion Results In Infertile Males Undergoing Assisted Reproductive Technology (ART)

A. Guney¹, A. Biricik², E. Tetik², H. Berkil², S. Kahraman³;
¹Marmara University School of Medicine, Istanbul, TURKEY, ²Istanbul Memorial Hospital, Reproductive Genetics Unit, Istanbul, TURKEY, ³Istanbul Memorial Hospital, Reproductive Endocrinology and ART Unit, Istanbul, TURKEY.

Infertility affects approximately 15% of all couples. Among these couples male factors are the aetiology in about 50% of cases. Besides many different factors in male infertility, chromosomal aberration and Y chromosome deletion screening became one of the standart procedures of ART clinics.
We report the cytogenetic and chromosome microdeletion results of 315 men with azoospermia or severe oligospermia. After clinical examination, seminal fluid and hormonal tests, cytogenetic investigation was performed by GTG-banding. The screening of microdeletions in 18 loci of the Y chromosome was performed by multiplex polymerase chain reaction. In 16 months we have performed 315 semen analysis according to WHO guidelines in 157 azoospermic (50%) and 158 oligospermic (50%) patients. Cytogenetic analysis was performed in 256 cases of the group and abnormality detected in 26 (10,2%) patients including 12 translocations, 8 Klinefelter syndromes, 3 invertions, 1 marker chromosome 15 carrier, 1 47,XYY, 1 mosaic 45,X(8%). Microdeletions of AZFa, AZFb, AZFc and AZFd regions of chromosome Y were detected in 1, 6, 11 and 11 patients respectively. Frequency of abnormal karyotype (10,2%) and frequency of Y microdeletions (4,1%) suggests the need for genetic testing of ART candidates and the requirement for genetic counselling.

L. Stuppia¹, A. Gaspari¹, V. Gatta¹, G. Calabrese¹, E. Morizio¹, D. Fantasia¹, I. Antonucci¹, R. Mingarelli², G. Palka¹;
¹Università "G. D'Annunzio", Chieti, ITALY, ²CSS Mendel Institute, Roma, ITALY.

About 10% of infertile males show microdeletions of the Y chromosome, the majority of which involving the AZFc locus. The DAZ gene maps within AZFc with four copies (DAZ1-4) and is considered as the major candidate for infertility. Since the PCR approach used for the screening of the Y chromosome can detect only deletions of all the DAZ copies, so far it is not know whether or not partial deletions of the cluster are related to a spermatogenesis failure. We studied the presence of partial deletions of the DAZ gene cluster in 42 infertile patients and in 67 fertile controls. This study was based on the presence within the DAZ sequence of three intronic SNPs able to distinguish among the four gene copies. The STSs sY581, sY586 and sY587 were PCR amplified and digested with Sau3A, Taq I and Dra I restriction enzymes, respectively. Absence of the band specific for DAZ2 was evidenced in 13 patients (30.9%) and in 10 controls (14.9%). Thus, the absence of DAZ2 appear compatible with normal spermatogenesis, but is two times more frequent in infertile males, suggesting that this condition could make patients prone to other genetic or environmental factor able to reduce their fertility. On the other hand, the loss of DAZ1-DAZ4 and DAZ2-DAZ3 was detected only in 4 patients, but never in controls, suggesting that this condition is not compatible with normal spermatogenesis. Experiments with Fiber FISH analysis are in progress in order to confirm these results with a different approach.

An Inherited Three Base Pair Deletion In A Sp1 Binding Site In The 5’ Non-coding Region Of Sry Gene Is Associated With Sex Reversal

J. G. Assumpção¹, A. T. Maciel-Guerra², A. P. Marques-de-Faria², M. P. De Mello¹;
¹Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP, BRAZIL, ²Dept. de Genética Médica, Universidade Estadual de Campinas, Campinas, SP, BRAZIL.

The condition named 46,XY pure gonadal dysgenesis is characterized by a female phenotype with full development of unambiguous female genitalia, normally developed Müllerian structures and streak gonads. Male to female sex reversal in 46,XY individuals results from failure of testis development and is presumed to be due to mutations in the SRY gene or in other genes involved in the sexual differentiation pathway. The majority SRY mutations described so far were found within the coding region, mainly in the HMG-box conserved domain. We have tested a female patient with 46,XY pure gonadal dysgenesis for mutations in the SRY gene. SRY complete open reading frame and 380 pb of the 5’ flanking region were amplified by PCR and directly sequenced. Sequence analysis revealed no mutations within SRY coding region. However, a three base pair deletion was found in the 5’ flanking sequence. This deletion involves a Sp1 binding site motif and is located next to a potential WT1 responsive element. This is the first report of a mutation in these putative SRY regulatory elements associated with sex reversal. Familial investigation revealed that the father bears the same deletion and two cousins were referred to have sexual ambiguity. Polymorphism was discarded, as it was not found in unrelated males. Different patient studied previously had the R30I familial SRY mutation within a phosphorylation site and it is associated to pure and partial gonadal dysgenesis as well as to normal phenotype. Those two mutations might influence sex determination depending on the in vivo environment.

The Leri-Weill and Turner-Syndrome homeobox gene SHOX encodes a cell-type specific transcriptional activator

R. J. Blaschke, E. Rao, A. Marchini, B. Niesler, M. Burnett, G. A. Rappold;
University Heidelberg, Heidelberg, GERMANY.

Functional impairment of the human homeobox gene SHOX causes short stature and Madelung deformity in Leri-Weill syndrome and has recently been implicated in additional skeletal malformations frequently observed in Turner syndrome. To enhance our understanding of the underlying mechanism of action, we have established a cell culture model consisting of four stably transfected cell lines and analysed the functional properties of the SHOX protein on a molecular level. Results show that the SHOX encoded protein is located exclusively within the nucleus of a variety of cell lines, including U2Os, HEK293, COS7 and NIH3T3 cells. In contrast to this cell-type independent nuclear translocation, the transactivating potential of the SHOX protein on different luciferase reporter constructs was observed only in the osteogenic cell line U2Os. Since C-terminally truncated forms of SHOX lead to Leri-Weill syndrome and idiopathic short stature, we have compared the activity of wild-type and truncated SHOX proteins. Interestingly, C-terminally truncated SHOX proteins are inactive with regards to target gene activation. These results for the first time provide an explanation of SHOX related phenotypes on a molecular level and suggest the existence of qualitative trait loci modulating SHOX activity in a cell-type specific manner.

M. Machatkova, S. Vilimova, I. Smetanova, M. Matejckova, A. Krebsova, M. Macek, Sr.;
Institute of Biology and Medical Genetics of the 2nd Medical School of Charles University and University Hospital Motol, Prague, CZECH REPUBLIC.

The aim of the study was the ascertainment of the Y chromosome microdeletions frequency in 197 Czech men with severe reproductive disorders, characterised by different types of abnormal sperm counts (SC) and morphology, referred for reproductive genetic counselling. For Y chromosome microdeletions detection multiplex PCR with amplification of STS markers for AZFa (sY84, sY86), AZFb (sY127, sY134), AZFc (sY254, sY255) and ZFY and SRY was used. Microdeletion frequency was analysed in four groups of males according to their sperm count: I. (azoospermia - 64x), II. (SC<1x106/ml - 19x), III. (SC 1-20x106/ml - 101x), IV. (SC>20x106/ml - 12x). The Y chromosome microdeletion was found in 8/197 (4.1%) examined males. Nevertheless, these microdeletions were detected in azoospermic males only in 7/64 (10.9%). The AZFc (sY254, sY255) deletion was disclosed in 4/64 (6.3%), AZFc (sY254, sY255) deletion combined with AZFb (sY127, sY134) deletion in 2/64 (3.1%) and AZFc (sY254, sY255) deletion combined with AZFb (sY134 only) deletion in 1/64 (1.6%). Microdeletion of AZFc (sY254, sY255) was also disclosed in one man referred with necrospermia without an exact sperm count. The 10.9% frequency of Y chromosome microdeletions in Czech azoospermic men does not differ from so far as reported range of its prevalence and no AZFa deletion was found.
The study was supported by grants: IGA 6462-3, IGA 6411-3, LN00A079, 111300003, 00000064203

N. Teran¹, T. Kunej¹, B. Zorn², B. Peterlin¹;
¹Division of Medical Genetics, Department of Obstetrics and Gynecology, University Medical Centre, Ljubljana, SLOVENIA, ²Andrology Centre, Department of Obstetrics and Gynecology, University Medical Centre, Ljubljana, SLOVENIA.

BACKGROUND. The clinical picture of trinucleotide repeat diseases: myotonic dystrophy type 1 (DM1), fragile XA syndrome (FraXA) and spinobulbar muscular atrophy (SBMA), is manifested as reduced infertility in men. The principle feature in adults with severe form of DM1 and SBMA is testicular atrophy, and for FraXA syndrome macroorchidism is characteristic. Several studies have reported that fertile males with a higher number of CAG repeats in normal range of androgen receptor have an increased risk of impaired spermatogenesis. In DM1 hypergonadotropic hypogonadism, leading to azoospermia and impotence, is frequently observed. An increased number of CTG repeat in infertile males could represent a risk of DM1 among offspring conceived by intracytoplasmic injection of sperm (ICSI).
The aim of this study was to compare the distribution of number of CTG repeats in normal range between infertile and fertile group of males to evaluate the involvement of CTG repeat number in male infertility.
PATIENTS. The number of CTG repeats in 107 infertile men (38 with azoospermia and 69 with oligoasthenoteratozoospermia), ICSI candidates, and in 102 fertile men with no sign of myotonia or muscle weakness was determined.
RESULTS. Among infertile males no premutation or mutation carriers were found. The distribution of the number of CTG repeats compared between infertile and fertile males was not statistically significant (p = 0.825).
CONCLUSION. We conclude that the number of CTG repeats in normal range does not contribute to male infertility. Moreover, this results show that increased risk of DM1 among offspring conceived by ICSI is unlikely.

Risk of sex chromosome mosaicism associated with Y chromosome AZFc microdeletions

P. C. Patsalis¹, C. Sismani¹, A. Ioulianos¹, C. Eftychi¹, C. Krausz², L. Quintana-Murci², K. McElreavey²;
¹The Cyprus Institute of Neurology and Genetics, Nicosia, CYPRUS, ²Institut Pasteur, Paris, FRANCE.

Deletions of specific Y chromosome regions cause male infertility. Recent advances in infertility treatment permits deleted Y-chromosomes to be transmitted to male offspring with the assumption that there will be no clinical consequences other than infertility in adult life. Recent data suggested that sex chromosome mosaicism could arise when AZFc-deleted Y chromosomes are transmitted from one generation to another by ICSI. If this hypothesis is correct, it raises the possibility that some patients with 45,X/46,XY karyotypes, such as patients with Turner-related phenotypes or sexual ambiguities may in fact harbour Y chromosome microdeletions. To test this hypothesis, we screened a group of patients with a 45,X/46,XY karyotype for Y microdeletions using several Y-specific markers. We show that 33% of patients presenting with Turner stigmata and/or sexual ambiguities and a 45,X/46,XY karyotype, carried Y chromosome microdeletions of distal Yq. These results highlight a potential risk for offspring born to fathers carrying Y chromosome microdeletions and treated by assisted reproductive techniques. The risk is the development of sex chromosome aneuploidy during foetal and embryonic development. The clinical consequences of this can be severe, including Turner syndrome, mixed gonadal dysgenesis, male pseudohermaphroditism and rarely mild mental retardation or autism. Our data may also explain in part the observation that in almost three-quarters of all 45,X patients with Turner syndrome, the X chromosome is maternal in origin. Concluding, the transmission of Y chromosome microdeletions could potentially have more severe clinical consequences other than male infertility, such as the development of sexual ambiguities and/or Turner stigmata.

A national based population screening for AZF deletions in patients with severe spermatogenic failure and normal fertile controls, using a specific study and experimental design

C. Sismani¹, A. Ioulianos¹, N. Fourouclas¹, T. Patroclou², C. Sergiou³, P. C. Patsalis¹;
¹The Cyprus Institute of Neurology and Genetics, Nicosia, CYPRUS, ²"Patroclou" Gynegologic & Obstetric Clinic, Limassol, CYPRUS, ³Fertility Center (CFC), Nicosia, CYPRUS.

Y chromosome microdeletions in the azoospermia factor (AZF) locus have been associated with spermatogenic failure. The frequency of AZF deletions is estimated to be about 7.3% in infertile men, although, in the literature the frequency varies between 1% and 55.5%. Therefore, considerable debate remains concerning the frequency, the position, and the phenotypes associated with Y chromosome microdeletions. In order to elucidate the above debate, we designed a national based population screening with well-defined study and experimental criteria. Eighty Greek-Cypriot patients that met the selection criteria were included in this study as well as 50 normal controls with proven fertility. All samples were collected from all districts of the island of Cyprus as the population is of the same religious, geographic and ethnic origin. All patients and controls had detailed clinical information and at least two semen analysis reports based on WHO standards. Samples with abnormal karyotypes, obstructive azoospermia or oligospermia with >2 X 106/ml were excluded from this study. The experimental design required a referral team and laboratory to undertake the responsibility, collect all samples, all clinical and laboratory information, isolate DNA and carry out all tests, data analysis and interpretation. This study showed that Y chromosome microdeletions are specific for spermatogenic failure. Under the specific patient selection criteria and experimental design the overall frequency is 6.3% or 12.5% among patients with azoospermia and 2.1% in oligospermia (<2 X 106/ml). The variation in deletion frequency reported in other studies is probably attributed to the patient selection criteria and experimental design.

Polymorphism of the MUC 1 mucin 60-bp microsatelite is not associated to women infertility nor to embryo implantation failure

L. R. Goulart¹, G. S. Vieira², J. Inacio², I. M. B. Goulart¹, L. R. Martelli³, J. G. Franco Jr⁴;
¹Universidade Federal de Uberlandia, Uberlandia-MG, BRAZIL, ²BioGenetics, Uberlandia-MG, BRAZIL, ³Universidade de Sao Paulo - FMRP-USP, Ribeirão Preto-SP, BRAZIL, ⁴Centro de Reprodução Humana - CRH, Ribeirão Preto-SP, BRAZIL.

Recent studies have demonstrated that the transmembrane mucin glycoprotein, Muc-1, is abundantly expressed at the apical surface of luminal epithelia under most conditions and is invariably reduced in receptive uteri. These and other observations have led to the suggestion that mucins serve an antiadhesive role and function to maintain a nonreceptive uterine state. A polymorphic variation of the MUC 1 gene (Horne et al., 2001 - Lancet, vol. 357, no. 9265) has been associated to women infertility due to suspected failure of embryo implantation, based on the significant greater size of the lower allele observed in the infertile group. Our aim was to confirm this preliminary data through a long-PCR methodology, based on primers flanking the 60-bp polymorphic microsatelite associated to the binding domain of the Muc-1 glycoprotein. DNA samples were obtained from twenty-one women patients, 10 fertile and 11 infertile (three or more implantation failures), and were amplified in long-PCR conditions using the Platinum Pfx DNA polymerase. Amplification generated fragments sizes from 1.6 to 2.9 kb. The average size for the lower allele was 1.68 kb for both groups, and for the upper allele was 2.29 and 2.47 kb (P>0.05), respectively for fertile and infertile groups. In conclusion, the 60-bp microsatelite polymorphism of MUC 1 gene was not associated to women implantation failure.

Y chromosome microdeletions in infertile azoo- and oligozoospermic men in Russia

V. Chernih, A. Chuhrova, L. Kurilo, A. Polyakov;
Research Center for Medical Genetics, Moscow, RUSSIAN FEDERATION.

Infertility affects 15% couples and in 50-60% of these cases qualitative and/or quantitative abnormalities of sperm are found. About in half of these cases the etiology of male infertility, defined as idiopathic, may be associated with genetic factors. Major known genetic cause for male infertility is Y-microdeletions in AZF locus (Azoospermia Factor) that contains three non-overlapping Yq11 regions (AZFa, AZFb and AZFc). Several genes, have been identified within these regions, are considered as candidates for male infertility. AZF-microdeletions are associated with spermatogenic failure ranging from Sertoli Cell Only Syndrome (SCOS) to spermatogenic arrest and severe hypospermatogenesis.
We have studied cohort of infertile men with azoospermia and severe oligozoospermia. Clinical examination, seminal fluid and cytogenetic analysis was carried out on 89 males. Cytogenetic investigation was performed according standard methods (GTG-banding). No chromosomal anomalies were found in men. Molecular analysis was carried out using leucocyte DNA by multiplex PCR. Primer set contains 9 Y-specific pairs: SRY; ZFY; sY84, sY86, sY615 (AZFa); sY127, sY134 (AZFb); sY254, sY255 (AZFc). Eight markers were selected on “Laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions” (Simoni et al., 2000), and one additional marker sY615 was used for AZFa subregion. Sequences of all primers are original. Microdeletions have been found in 13 cases. Eleven patients had microdeletions in AZFc (9 with azoospermia and two - severe oligozoospermia), 2 patients had microdeletions AZFb+c and azoospremia. The frequency of microdeletions in this study is 14.6% that is consistent with published data.

G. Pilia¹, L. Crisponi¹, M. Uda¹, F. Chiappe², M. Deiana¹, G. Usala¹, P. Amati³, D. Bonneau³, F. Faravelli⁴, J. Tolmie⁵, L. Bisceglia⁶, M. R. Piemontese⁶, L. Zelante⁶, A. Iolascon⁷, P. Gasparini⁶, G. Crisponi², L. Boccone⁸, A. Percesepe⁹, A. Forabosco⁹, G. Monni⁸, S. Loche⁸, A. Cao¹;
¹CNR, IRTAM, Cagliari, ITALY, ²University of Cagliari, Cagliari, ITALY, ³Service de Genetique Medicale, Poitiers, FRANCE, ⁴Ospedale Galliera, Genova, ITALY, ⁵Yorkhill NHS Trust, Glasgow, UNITED KINGDOM, ⁶IRCCS, Ospedale CSS, San Giovanni Rotondo, ITALY, ⁷University of Bari, Bari, ITALY, ⁸Ospedale Microcitemico, Cagliari, ITALY, ⁹University of Modena, Modena, ITALY.

Type I Blepharophimosis/Ptosis/Epicanthus inversus Syndrome (BPES) is an autosomal dominant disorder in which eyelid abnormalities are associated with Ovarian Failure leading to Female Infertility. Apparently, type II shows only the eyelid defects . We recently cloned a novel gene, FOXL2, belonging to the family of the winged helix/forkhead transcription factor, that we found to be mutated in both types of BPES.
We carried out a FOXL2 mutation screening on 45 BPES families. On all 13 type I BPES families analyzed we identified nonsense and frameshift mutations. In 7 type II BPES patients we observed apparently milder mutations: a 30 bp in-frame duplication (909_939dup) present in 3 independent cases, 3 different missense mutations and a 3 basepair deletion. We detected FOXL2 mutations in 7 additional BPES patients whose clinical type could not be determined. These results suggest a genotype-phenotype correlation ; FOXL2 loss-of-function mutations are probably incompatible with any kind of female fertility while milder mutations could allow a child-bearing activity but nonetheless affect ovarian function. For example, the 909_939dup was present in a girl with overt Ovarian Failure whose affected mother also had a history of Premature Ovarian Failure (POF) . A position effect for the FOXL2 gene or mutations in another gene could be responsible for those cases in which we found no mutations. In addition, we are concluding the screening of FOXL2 for 100 POF patients, and to now we only found one missense mutation in a patient in the coding region of the gene.

R. Dada, N. Gupta, K. Kucheria;
All India Institute of Medical Sciences, New Delhi, INDIA.

Microdeletion of the long arm of the Y chromosome are associated with spermatogenic failure and have been used to define three regions on Yq (AZFa, AZFb and AZFc) which are critical for spermatogenesis and are recurrently deleted in infertile males.
102 infertile males were included in this study. Semen analysis was done in each case to determine the spermatogenic status. They were subjected to detailed clinical examination, endocrinological and cytogenetic study.In all cytogenetically normal cases (n=70) microdeletion analysis was carried out using PCR. For this genomic DNA was extracted using peripheral blood. The STS primers tested on each subject were sY84, sY86 (AZFa); sY127, sY134 (AZFb); sY254, sY255 (AZFc).Eight of the seventy cases (11.4%) showed deletion of at least one of the STS markers. Four cases had AZFc deletion, three cases had AZFa and AZFb deletion and one case showed AZFb deletion alone. The 3 cases with AZFa and AZFb deletions had Sertoli Cell Only Syndrome (SCO). Two of the three cases with AZFc deletion had hypospermatogenesis and in the third case there was maturation arrest. In two cases with AZF deletion FNAC was not possible as they were cryptorchid. Thus cases with AZFa and AZFb deletion have poor prognosis and cases with AZFc deletion can go in for Assisted Reproductive technology as there testis show spermatogenesis though at a reduced rate. Therefore in all cases of idiopathic infertility a genetic aetiolgy should be established to provide the most adapted therapeutics to the infertile couple.